Media used to grow cultured cells for both industrial and clinical applications are usually chemically undefined or, at best, semi-defined. It would be advantageous to employ chemically-defined media in cell culture systems; however, most attempts to grow cultured cells exclusively in chemically-defined media have been unsuccessful and result in a high frequency of cell death and/or poor cell performance. These efforts have failed, at least in part, because those components of undefined media that promote cell viability and performance remain uncharacterized. Thus, it would be desirable to develop improved methods of identifying media components that support and enhance cell viability and performance.
Hydrolysates are the most common undefined substance used in bacteriological media today for both clinical and fermentation applications. Current media optimization frequently starts with these undefined substances. Hydrolysates are used to replace serum (another undefined substance) in mammalian culture (Saha and Sen, (1989) Acta Virol. 33:338).
The inclusion of tissue and protein hydrolysates in bacterial growth media has been practiced since the late 1800s. Retger, (1927) J. Immunology 13:323, provided some of the earliest details on preparing and combining hydrolysates. Retger's data demonstrated that best toxin yields from Corynebacteria diptheriae were obtained with medium formulated with hydrolysates containing a lower percentage of full-length proteins and a higher concentration of peptides. Kihara et al., (1952) J. Biol. Chem. 197:801, also found that cultured cells performed better in medium containing peptides as compared with the constituent amino acids.
There are several drawbacks associated with using digests in cell culture media, for example, the range of peptide sequences available for incorporation into culture media is limited by the starting substrate and enzyme or acid used in the digestion. Many of the currently-available digests are difficult to reproduce, with significant lot-to-lot variation being commonplace. Digests of tissue obtained from a slaughterhouse are often used as components of cell culture media. These digests are among the most difficult to reproduce since the starting material and ratios of the starting substrate vary. Casein digests are more reproducible because milk is a more homogenous source of protein than slaughterhouse tissue. However, the range of peptide sequences generated by an enzymatic or acid digestion of casein is small. Most media manufacturers blend these two types of digest to yield a medium that provides better results than can be achieved with either digest used alone. Unfortunately, the use of blends adds another level of complexity to the manufacturing process and is also a source of lot-to-lot variability.
Moreover, both hydrolysates and sera are problematic for pharmaceutical applications, since each potentially harbors pathogens. The undefined nature of hydrolysates and sera also leads to problems in manufacturing. To illustrate, the high molecular weight components found in both types of undefined substances create additional downstream processing costs. Furthermore, the undefined nature of hydrolysates and sera leads to lot-to-lot variability. Previous attempts at developing chemically-defined media, however, have generally suffered from sub-optimal cell performance and unacceptable levels of cell mortality.
Historically, there have been several approaches employed to determine the type(s) of nutrients consumed or preferred by cells grown in culture. One of the most common practices is post-culture analysis, whereby the spent medium is evaluated to identify constituents removed from the medium. This approach has only rarely lead to the identification of compounds that can be isolated for use as a medium component or can be employed as a benchmark to monitor future hydrolysate or serum lots. In addition, this approach cannot identify compounds that function through signaling and are not physically removed from the medium.
Zhao et al., (1996) Appl. Microbiol. Biotechnol. 45:778, compared the bacterial growth-stimulating activity of bovine hemoglobin with that of specific peptides from a peptic hydrolysate of this protein. A particular peptide fragment was demonstrated to promote cell growth of gram negative bacteria to a greater extent than the intact protein. No such enhancement was observed when the constituent amino acids of this active fragment were assayed, leading these investigators to suggest that this peptide fragment did not simply act as an amino acid supplier, but rather interacted with peptide permeases on the cell membrane. This strategy can be employed in other systems as well to provide some information as to specific biologically-active peptides produced by hydrolysis of whole proteins.
More recently, Automated Cell Technologies (ACT; Pittsburgh, Pa.) has suggested that it will use a “combinatorial cell culture” to discover improved media for growing hematopoietic stem cells ex vivo. In Vivo: Bus. Med. Rep. 15:38 (December 1997). Utilizing a 384-well microtiter plate and a robotic pipetting system, ACT proposes to add different growth factor combinations to various mixes of culture media in an effort to identify a culture medium that will support stem cell growth ex vivo. It is unstated whether chemically-defined or undefined media will be used.
All of the previously-described methods fail to further an understanding of the physical, chemical or other properties of the medium components that contribute to the enhanced cell performance in culture. As a result, these methods are inefficient as they fail to provide a means of predicting and systematically screening additional lead compounds.
Most of the research on quantitative structure-activity relationships (QSAR) have focused on small organic molecules in medicinal and environmental chemistry. Peptides have not been studied as much owing to difficulties in developing descriptors for amino acid side chains. The earliest attempt to quantify amino acids for QSAR was by Sneath, (1966) J. Theoretical Biology, 12:157, who developed four semi-quantitative descriptors for amino acids. Later, Hellberg et al., (1987) J. Med. Chem. 30:1126, developed a set of principal components, which were derived from twenty-nine measured and theoretical properties of amino acids, including molecular weight, isoelectric point, nuclear magnetic resonance parameters, logP (hydrophobicity), thin layer chromatography, and high performance liquid chromatography parameters. Principle component analysis led to three principal components, which Hellberg et al. called z1, z2 and z3. Theoretically-derived parameters have been developed by Norinder, (1991) Peptides, 12:1223, and Cocchi and Johansson, (1993) Quant. Struct.-Act. Relat. 12:1. In all of these instances, parameters were developed for the individual amino acids, but none were measured on whole peptides.
Cho et al., (1998) J. Chem. Inf. Comput. Sci. 38:259, used a rational drug design approach to identify peptides in a targeted virtual library having bradykinin-potentiating activity. This group identified virtual peptides predicted to be enhanced in amino acid building blocks with bradykinin-like activity based on analysis of a starting set of peptides known to possess such activity. These investigators employed topological indices or physiochemical descriptors of individual amino acid building blocks from known leads to design a virtual targeted library. Cho et al. also report that computational limitations prevented complete analysis of all indices for every virtual peptide within their targeted library. Cho et al. did not apply their methods to media development.
Thus, previous attempts to improve culture media have largely relied on ad hoc trial-and-error techniques. There remains a need in the art for systematic and predictive methods for identifying medium components to improve cell performance in culture. Moreover, there is a need in the art for high through-put methods for identifying medium components.